direct bioconversion of cellulose into ethanol by mixed ... · direct bioconversion of cellulose...

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International Journal of Innovative Research in Science,

Engineering and Technology

(An ISO 3297: 2007 Certified Organization)

Vol. 4, Issue 4, April 2015

Copyright to IJIRSET DOI: 10.15680/IJIRSET.2015.0404010 1870

Direct Bioconversion of Cellulose into Ethanol

by Mixed Culture –Batch Kineticand

Modeling

P.Nagarajan1,2

, T.Karunanithi3, Yulin Deng

4

Research Scholar, School of Chemical & Biomolecular Engineering and IPST at Georgia Tech, Georgia Institute of

Technology, Atlanta, Georgia ,USA1.

Associate Professor, Department of Chemical Engineering, Annamalai University, India2.

Professor (Rtd) , Department of Chemical Engineering , Annamalai University , India3.

Professor, School of Chemical & Biomolecular Engineering and IPST at Georgia Tech, Georgia Institute of

Technology , Atlanta, Georgia ,USA4.

ABSTRACT:The present work attempts to investigate the ability of fusariumoxysporum and neurosporacrassa to

ferment cellulose to ethanol directly. Batch experiments were performed for both the strain at 30.c in 250ml conical

flasks.1000ml of production medium was distributed equally in 7 sterile,250 ml conical flasks, each containing various

initial concentrations of cellulose 20,30,40,50,60,70 and 80g/l respectively.20ml of inoculum medium was transferred

to each 100ml production medium in sterile conditions. Initial values of ethanol concentration, cellulose concentration

and cell mass were determined. The inoculated flasks were maintained at 300 C in a rotary shaker operating at 200 rev

min-1

.Analyses were carried out for every 24 hours using the contents of the various conical flasks.Experiments were

conducted for mixed cultures of different compositions 80:20, 60:40, 40:60, and 20:80by volume respectively.The

cultures fusariumoxysporum and neurosporacrassa were mixed in proportion 80:20,60:40,40:60,and 20:80 respectively

mixed cultures were used for inoculation.Based on the kinetic models, equations have been developed for the

determination cell mass, product and substrate concentration as a function of time. Using the experimental data,

constants of these equations have been evaluated. These mathematical relationships can be used with reasonable

accuracy.

KEYWORDS: Cellulose, Ethanol, Mixed culture, Batch processes and Kinetic Studies.

I. INTRODUCTION

Direct bioconversion of cellulose to ethanol is a process in which the same microorganism carries out both hydrolysis

and fermentation of cellulose to ethanol in one operation. Simultaneous hydrolysis of cellulose to ethanol improves the

kinetics and economics of biomass conversion by minimizing accumulation of hydrolysis products that are inhibitory.

Only limited work has been carried out in this area of research.

Fusariumoxysporumhas been reported to ferment cellulose to ethanol in a one step process. But not much detail on

yields has been published. The filamentous fungus FusariumOxysporum is known for its stability to produce ethanol by

simultaneous saccharification and fermentation of cellulose. The main disadvantage is the slow conversion rate when

compared with yeast.

Neurosporacrassa species have not been extensively studied from a biotechnological viewpoint for their cellulolytic

activity or ethanol production till now. However, the production of cellulaseas well as aryl - D-glucosidase from

Neurosporacrassahas been reported. Neurosporacrassa, excreting cellulose and xylanase in the culture fluid, ferments

cellulose directly to ethanol. In view of its ability to produce ethanol through termination of pentose sugars as well as

D-glucose and cellulose, Neurosporacrassa strains merit further detailed study.

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It is possible that when mixed cultures of two microorganisms are used, both species may grow faster than they do

separately. Such an interaction called mutualism may help increase the yield of the product.The results are presented

for the systematic analysis of the influence of various parameters that control substrate utilization, microbial growth

and product formation.The models developed for the bioconversion of cellulose. Riccati type logistic equation has been

used for the prediction of cell mass. Leudeking-Piret model is incorporated with logistic equation to get the product

concentration. Modified Leudeking model is used for the estimation of substrate concentration.

The experimental data are analyzed for the effect of parameters that influence the process. The concentration profiles

obtained from the models developed are compared with the experimental results and adequacy and limitations of these

models are discussed in detail. Correlations have been developed for the prediction of the values of the model

parameters.

II. LITERATURE SURVEY

Cellulose, a major component of the cell wall of plants, is the most abundant and renewable carbohydrate. In recent

years, it has been proposed that waste cellulosic biomass could be used as a cheap and readily available sugar to replace

starchy materials in fermentation (Bailey and Ollis, 1986).

The energy and environmental gains from cellulosic ethanol, known as bioethanol, will be substantial .Bioethanol

represents a net energy gain of 60,000 Btu per gallon, up to three times more than starch ethanol. Ultimately, large-

scale cellulosic ethanol production will require production of dedicated crops such as switch grass or fast-growing

poplar trees. CO2 will cycle in and out of the atmosphere as ethanol is burned and new crop rotations are planted (Lynd

et al., 2002).

Bioconversion of cellulosic materials to ethanol has attracted world-wide interest in recent years. This process is

generally accomplished in two steps by conventional methods. In the first step, the biopolymers are hydrolyzed to

monosaccharides. In the second step, these monosaccharides are converted to ethanol (Mielenz, 2001).

Have investigated the intracellular metabolite profiling of Fusariumoxysporumconverting glucose and glucose-xylose

mixture ( Panagiotou et al., 2005).

Several filamentous fungi have been reported to directly ferment cellulose to ethanol, though on rich, undefined media.

These include members of the genera Aspergillus, Rhizopus,Monilia ,Neurosporacrassa(Deshpande et al., 1986), and

Fusariumoxysporum.The recombinant Klebsiellaoxytoca SZ21 developed by (Zhou et al., 2001) was able to directly

produce ethanol from amorphous cellulose, although with insufficient ethanol yield.Ethanol production was not

affected when the activity of the former two enzymes was varied within a wide range as reported by(Christakopoulos et

al, 1989).

During the different phases of the cultivations, the intracellular profiles were determined under aerobic and anaerobic

conditions( Panagiotouet al., 2004).

(Aditya et al., 1990) have discussed the production of ethanol from sugars in wood hydrolysate. Wood hydrolysate used

for ethanol production by two strains of Fusariumoxysporum contained 2.3% (w/v) reducing sugars - xylose and

glucose (Kadam et al., 2000).

Neurosporacrassa, aerobic fungi, has a potential role in producing ethanol from biomass. This organism has an in

durable system for the repair of genetic damage (Ikram et al., 2003).

The corresponding ethanol concentrations in the fermentation medium were 4.6% and 6.4%. These results clearly

demonstrated that a large portion of insoluble carbohydrates from sorghum was converted by simultaneous

saccharification and fermentation to ethanol (Lezinov et al., 1994). No studies on mixed cultures involving

FusariumoxysporumandNeurosporacrassa have been reported so far.

III. METHODS AND EXPERIMENT

All bioconversion experimental investigations require periodic subculture and maintenance of microorganism,

preparation of media and inoculums, careful conduction of experiment under aseptic condition besides others. The

laboratory strains (wild type) of Fusariumoxysporum MTCC 739 and Neurosporacrassa MTCC 839 .The basic

requirement of the medium was obtained from the literature (Christakopoulos et al., 1989; Deshpande et al., 1986).

A :Growth medium

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The composition of growth medium for Fusariumoxysporum is as follows: potato- 200 g/l; sucrose- 20 g/l and

agar- 20 g/l. The pH of the medium was adjusted to 6.5.The composition of growth medium for Neurosporacrassa is as

follows: Sucrose- 20.0 g/l;KH2PO4(anhydrous) - 5.0 g/l;Tri-sodium citrate-2.5g/l; MgSo4.7H2O- 0.2 g/l; NH4NO3

(Anhydrous)- 2.0 g/l;CaCl2.2H2O- 0.1 g/l; Biotin solution (5 mg/ml)- 1.0 ml, Trace element solution 1 ml and agar -

20.0 g/l.

The trace element solution was prepared by dissolving in 95 ml of water: Citric acid- 5.0g; ZnSO4.7H2O -5.0

g; Fe (NH4)2(SO4)2.6H2O -1.0g; CuSO4.5H2O- 0.25g; MnSO4 4H2O and H3BO3 (anhydrous) added in trace so that the

total volume of trace element solution is about 100ml. The stock culture for both the strains were maintained on a

sterile medium and incubated for a period over 7 days for Fusariumoxysporumand 3 days for Neurosporacrassa at a

temperature of 30C and then stored in a refrigerator at 0C for further use.

The strain used for this study is shown in Figs 1&2.

B: Preparation of mixed culture broth

From the mutant strains of Fusariumoxysporum and Neurosporacrassa, mixed culture broths were prepared at different

proportions of Fusariumoxysporum and Neurosporacrassa in the ratio of 80:20, 60:40, 40:60, and 20:80 by volume

respectively. They were kept in the incubator and were incubated at 30 C .

C: Production Medium

The production medium for Fusariumoxysporum is as follows (Mishra et al. 1984; Basil Macris, 1984): KH2PO4- 2

g/l; ZnSO4.7H2O- 0.02 g/l; Mg SO4-0.3 g/l;FeSO4.7H2O- 0.05 g/l;CaCl2 - 0.3g/l; MnSO4.4H2O- 0.02 g/l; Peptone -5g/l;

Malt extract- 3g/l; Yeast extract- 3 g/l and CoCl2-2 g/l.The production medium for Neurosporacrassa is as follows:

Malt extracts - 3 g/l; Yeast extract - 3 g/l and Peptone -5 g/l.

Fig. 1 Scanning Electron Micrograph (SEM) of Neurosporacrassa cell grown in solid medium

Fig. 2 Scanning Electron Micrograph (SEM) of oxysporum cell grown in solid medium

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D:Assay techniques

Estimation of cell mass

Centrifuge tubes were well washed and dried in an oven to remove all the moisture. Weights of empty dry centrifuge

tubes were found out using electronic balance. 10ml of the broth was taken in the centrifuge tube and centrifuged for

20min. The settled biomass was made free of water and it was kept in the oven to remove all moisture. The weights of

centrifuge tubes with the biomass were found out by electronic balance. The weight of the cell mass was found from

the difference in measured weights.

Estimation of cellulose by Anthrone reagent method

The estimation of cellulose was carried out using spectrophotometer. 3ml of acetic reagent prepared by mixing 150ml

of 80% acetic acid and 15ml of concentrated nitric acid was added to one gram of the sample in a test tube and mixed

in a vortex mixture. The tube was placed in a water bath at 100°C for 30 minutes, cooled and then centrifuged for 15-20

minutes. The supernatant liquid was discarded; the residue was washed with distilled water. Then 10ml of 67%

sulphuric acid was added and allowed to stand for 1 hour. One ml from the above solution was diluted to100ml. To one

ml of this diluted solution, 10ml of anthrone reagent, prepared by dissolving 200mg anthrone in 100ml concentrated

sulphuric acid and chilled for 2 hours before use, was added and mixed well. The tubes were heated in boiling water

bath for 10 minutes, cooled and the intensity of color was measured at 630 nm in a spectrophotometer. The amount of

cellulose in the sample was estimated from the standard chart prepared by using known cellulose concentration

(Sadasivam and Manickam 1996).

Estimation of Ethanol

The concentration of ethanol was determined using gas chromatograph containing FID. Poropak column by which all

the hydrocarbons can be determined was used for this purpose. The carrier gases (nitrogen) and fuel gas (hydrogen and

oxygen) were regulated at a certain pressure. Flame was ignited at the flame ionization detector port. The injector,

detector and oven temperatures were programmed. After reaching the stability, when the oven temperature and

detector, injector temperature were at the programmed temperature, a sample was injected from fermentation flask into

injector port by using a micro syringe at 2 psi pressure. The peak eluted was noted. By knowing, area of peak, the

concentration of ethanol was calculated using standard graph.

IV RESULT AND DISCUSSION

A: Pure wild cultures

The pure cultures of Fusariumoxysporum and Neurosporacrassa are compared in terms of cell growth, ethanol

production and cellulose conversion. It should be kept in mind that the ultimate aim is to select systems that yield

maximum alcohol.

Both Fusariumoxysporum and Neurosporacrassa yield ethanol in significant amounts. However, Fusariumoxysporum is

more efficient in the production of ethanol when compared to Neurosporacrassa. In fact ethanol yield for

Fusariumoxysporum is 15% higher than that of Neurosporacrassa (Figs 3b and 4b). Tis is true for all initial

concentrations of cellulose considered for this study. It is also noted that as the initial concentration of cellulose is

raised, the alcohol concentration in the broth also increases.

Cell mass production is high for Neurosporacrassacompared to Fusariumoxysporum. Neurosporacrassa produces 7%

more cell mass than Fusariumoxysporum. (Figs 3a and 4a).

Cellulose conversion, on an average, is 76% for Fusariumoxysporum and 79% for Neurosporacrassa indicating an

increase of 3% for Neurosporacrassa (Figs 3c and 4c.) . These points considered together indicate that

Neurosporacrassa produce more by products. Hence the ethanol production efficiency decreases.

B:Effect of Initial concentration:

Figs 3 and 4 indicate the effect of initial concentration on the performance of the pure cultures. As the initial

concentration of cellulose is varied cell mass production varies proportionally. Higher cell mass concentration is

obtained for 80% and the least in for 20%. The trend is same for both the pure cultures of Fusariumoxysporum and

Neurosporacrassa.The results clearly indicate that the more the initial concentration of cellulose the higher the

production of ethanol.

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(a) (b)

( a) (b)

(c)

(a) (b)

(C)

C: Wild mixed cultures

One of the main objectives of the present work is to verify whether mixed cultures are more effective than the pure

cultures. Mixed cultures of various ratios of Fusariumoxysporum and Neurosporacrassa are compared with the wild

(c) time, hr time, hr

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time, hr Fig.4 Effect of Initial Concentration on Nerosporacrassa.-a.Cellmass, b.Ethanolc.Cellulose

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Fig.3Effect of Initial Concentration on Fusariumoxysporuma.Cellmass, b.Ethanolc.Cellulose

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cultures in terms of cell growth, ethanol production and cellulose conversion. This is done to determine the proper

composition of the microorganisms in the mixed cultures that yield maximum alcohol (Table I).

Cell mass:

The wild culture of Fusariumoxysporum and the mixed culture having the composition 80% Fusariumoxysporum and

20% Neurosporacrassa(FO80 - NC20) have nearly same cell mass concentration at the stationary phase even though

the initial rate of generation of cell mass is high for mixed culture. The addition of Neurosporacrassa into the mixture

(FO60-NC40, FO 40-NC60 and FO20-NC80) reduces the final cell mass concentration. In fact, in all these three cases

the final cell mass concentration is less than the concentration of pure cultures.

Ethanol:

The mixed culture (FO80 - NC20) yields maximum alcohol when compared to other cultures. It is followed by FO60-

NC40, pure culture Fusariumoxysporum, FO40-NC60 and the pure culture Neurosporacrassa. FO20-NC80 gives least

alcohol when compared to all others and hence need not be used for fermentation studies. FO60-NC40 is slightly more

efficient than pure culture of Fusariumoxysporum.

Cellulose:

For low initial concentrations of cellulose, the conversion by (FO80 - NC20) is high when compared to others. Higher

than this initial concentration results in the decrease of conversion. There is only marginal difference among the various

TABLE I

FERMENTATION OF CELLULOSE TO ETHANOL BY MIXED CULTURE

Mixture culture

ISC

(g/l)

Time

(hr)

FO 80% NC 20% FO 60% NC 40% FO 40% NC 60% FO 20% NC 80%

X

g/l

P

g/l

C

g/l

X

g/l

P

g/l

C

g/l

X

g/l

P

g/l

C

g/l

X

g/l

P

g/l

C

g/l

000 0.79 0.00 00.20 0.79 0.00 00.20 0.79 0.00 00.20 0.79 0.00 00.20

024 2.08 1.78 15.59 1.49 1.47 16.40 1.95 0.81 17.01 1.38 0.45 18.25

048 2.78 3.39 11.91 2.78 2.72 13.10 2.75 1.86 13.45 2.19 1.21 14.94

072 3.21 4.29 10.38 3.52 3.39 11.75 03.2 2.53 11.62 2.93 1.83 12.54

096 3.35 5.05 09.25 4.08 3.94 10.88 3.39 3.01 10.60 3.20 2.29 11.06

20 120 3.48 5.79 08.19 4.61 4.46 10.11 3.46 3.45 09.81 3.28 2.67 10.06

144 3.57 6.54 06.85 5.14 4.99 09.34 3.48 3.87 09.07 3.33 3.01 09.26

168 3.62 7.28 05.74 5.66 5.52 08.45 3.52 4.29 08.35 3.35 3.35 08.01

192 3.70 8.02 04.68 6.19 6.04 07.65 3.53 4.71 07.22 3.37 3.78 07.11

216 3.74 8.86 03.65 6.71 6.67 06.74 3.54 5.32 06.59 3.38 4.11 06.27

240 3.79 9.62 02.25 7.24 7.19 05.89 3.54 5.74 05.89 3.39 4.46 05.55

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TABLE :I(cond.)

000 0.79 0.00 00.30 0.79 0.00 00.30 0.79 0.00 00.30 0.79 0.00 00.30

024 2.10 1.84 24.79 1.99 1.30 24.34 1.98 0.86 25.26 1.40 0.47 26.44

048 2.95 3.54 20.15 2.68 2.63 18.94 2.76 2.02 19.45 2.26 1.26 21.18

072 3.34 4.49 18.22 3.04 3.38 16.57 3.33 2.77 16.35 2.85 1.91 17.29

096 3.55 5.28 16.83 3.29 3.95 15.11 3.51 3.31 14.62 3.20 2.39 14.85

30 120 3.68 6.06 15.51 3.42 4.50 13.78 3.60 3.78 13.31 3.28 2.79 13.15

144 3.79 6.83 14.20 3.52 5.03 12.52 3.70 4.24 12.08 3.32 3.14 11.82

168 3.85 7.60 12.65 3.63 5.57 11.11 3.80 4.69 10.90 3.39 3.49 10.54

192 3.92 8.37 11.35 3.68 6.10 09.56 3.82 5.15 09.25 3.45 3.88 09.20

216 3.94 9.26 09.65 3.70 6.79 08.25 3.83 5.81 08.11 3.48 4.27 07.68

240 3.94 10.11 08.34 3.70 7.34 06.85 3.83 6.27 06.86 3.49 4.63 06.35

z 0.79 0.00 00.40 0.79 0.00 00.40 0.79 0.00 00.40 0.79 0.00 00.40

024 2.15 1.90 32.67 2.02 1.34 32.48 1.99 0.88 33.30 1.42 0.47 34.85

048 3.01 3.74 25.93 2.87 2.77 24.90 2.84 2.08 25.32 2.17 1.28 27.51

072 3.55 4.75 23.15 3.32 3.58 21.58 3.37 2.87 20.98 2.92 1.96 22.01

096 3.85 5.60 21.15 3.46 4.19 19.57 3.55 3.42 18.59 3.28 2.47 18.54

40 120 3.98 6.42 19.29 3.61 4.76 17.81 3.62 3.91 16.75 3.39 2.87 16.23

144 4.05 7.23 17.45 3.72 5.32 16.12 3.68 4.39 15.00 3.47 3.24 14.32

168 4.12 8.05 15.23 3.82 5.89 13.98 3.72 4.85 13.36 3.55 3.60 12.57

192 4.15 8.87 13.26 3.88 6.45 12.34 3.85 5.32 11.23 3.56 3.95 10.55

216 4.17 9.78 11.24 3.90 7.18 10.54 3.88 5.99 09.67 3.59 4.39 08.85

240 4.17 10.69 09.23 3.90 7.76 09.12 3.92 6.47 07.95 3.61 4.75 07.01

ISC Time

(g/l) (hr)


X

g/l

P

g/l

C

g/l

X

g/l

P

g/l

C

g/l

X

g/l

P

g/l

C

g/l

X

g/l

P

g/l

C

g/l

000 0.79 00.00 00.50 0.79 0.00 00.50 0.79 0.00 00.50 0.79 0.00 00.50

024 2.50 02.06 41.68 2.05 1.41 41.15 2.35 0.93 42.41 1.45 0.50 44.11

048 3.79 04.14 33.17 3.24 2.97 31.67 3.55 2.22 32.28 2.36 1.34 34.99

072 4.22 05.28 29.61 3.82 3.85 27.43 4.01 3.08 26.66 3.10 2.06 27.99

096 4.32 06.20 27.18 3.95 4.50 24.90 4.08 3.68 23.63 3.45 2.59 23.64

50 120 4.41 07.10 24.85 4.02 5.11 22.69 4.13 4.20 21.32 3.58 3.02 20.70

144 4.51 08.00 22.54 4.05 5.72 20.51 4.18 4.70 19.20 3.62 3.40 18.41

168 4.55 08.89 19.85 4.09 6.32 18.37 4.19 5.20 16.95 3.68 3.77 16.31

192 4.56 09.79 17.25 4.15 6.92 15.98 4.20 5.70 14.73 3.73 4.14 13.85

216 4.57 10.78 14.95 4.16 7.69 13.72 4.21 6.41 12.11 3.74 4.59 11.56

240 4.57 11.72 12.74 4.16 8.29 10.95 4.21 6.92 10.21 3.75 4.97 09.58

000 0.79 00.00 00.60 0.79 0.00 00.60 0.79 0.00 00.60 0.79 0.00 00.60

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TABLE:I(cond.)

024 2.25 02.08 49.27 2.07 1.52 49.52 2.03 0.97 50.94 1.48 0.50 53.40

048 3.90 04.31 38.61 3.53 3.31 36.56 3.53 2.37 38.31 2.26 1.36 42.62

072 4.24 05.51 34.21 4.23 4.32 30.78 4.25 3.31 31.15 3.05 2.11 34.17

096 4.41 06.48 31.17 4.35 5.06 27.59 4.35 3.95 27.33 3.45 2.66 28.85

60 120 4.57 07.42 28.29 4.47 5.74 24.91 4.40 4.51 24.47 3.62 3.10 25.32

144 4.65 08.36 25.44 4.52 6.41 22.33 4.41 5.05 21.86 3.67 3.50 22.40

168 4.71 09.30 22.12 4.58 7.08 19.45 4.44 5.59 19.29 3.76 3.88 19.82

192 4.75 10.23 19.23 4.61 7.75 16.74 4.47 6.12 16.12 3.82 4.29 16.59

216 4.79 11.34 16.12 4.61 8.61 13.52 4.49 6.89 13.24 3.83 4.73 13.49

240 4.79 12.32 13.45 4.62 9.28 10.65 4.49 7.42 11.14 3.85 5.12 11.15

ISC Time

(g/l) (hr)


X

g/l

P

g/l

C

g/l

X

g/l

P

g/l

C

g/l

X

g/l

P

g/l

C

g/l

X

g/l

P

g/l

C

g/l

000 0.79 00.00 00.70 0.79 0.00 00.70 0.79 0.00 00.70 0.79 0.00 00.70

024 2.39 02.22 58.53 2.12 1.56 58.61 2.55 0.99 59.97 1.50 0.51 61.57

048 3.99 04.74 45.75 3.93 3.59 43.86 4.10 2.47 45.55 2.32 1.40 48.21

072 4.87 06.10 40.39 4.69 4.74 37.11 4.44 3.47 37.20 3.14 2.18 37.70

096 5.01 07.17 36.80 4.85 5.56 33.33 4.53 4.15 32.62 3.56 2.76 31.30

70 120 5.12 08.21 33.42 4.93 6.31 30.14 4.58 4.74 29.14 3.69 3.22 27.08

144 5.19 09.24 30.10 4.97 7.05 27.05 4.57 5.31 25.93 3.78 3.63 23.88

168 5.21 10.27 26.45 5.02 7.79 22.56 4.65 5.86 22.88 3.87 4.08 20.53

192 5.26 11.30 22.84 5.08 8.52 19.65 4.69 6.42 19.45 3.93 4.45 17.52

216 5.28 12.52 19.45 5.09 9.45 16.35 4.70 7.22 16.12 3.98 4.90 14.32

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IV. KINETIC MODELING

Each individual cell is a complicated multicomponent system, which is frequently not spatially homogenous even at the

single cell level. Many independent chemical reactions occur simultaneously in each cell. In a growing cell population,

there will be significant cell to cell heterogeneity. There will be age distribution of the cells. Cells of different ages are

characterized by different types of metabolic functions and activities (Bailey and Oills, 1986).

This leads to the unstructured model.In the present study, the mass transfer effects are lumped together with the

biochemical reactions and the rate expressions are represented in terms of the gross properties of the system. Thus the

concentrations represent the conditions at the bulk only.Models are described to represent change in concentrations of

cell mass, product and substrate in the bulk as a function of time.

A: Microbial growth Logistic curve

Rate of growth of cell is proportional to the cell mass concentration present at that time(x). The rate will stop when the

cell mass concentration reaches stationary phase. When the cell mass concentration is near the stationary phase rate will

slow down. That is, growth rate also depends on how far the cell mass concentration at a given point of time is away

from the cell mass concentration at stationary phase.

kdtx)β(1xk

dx

-------------- (1)

The above Riccati equation which can be integrated to give the logistic curve equation.

)e(1)x/x(1

exx

kt

s0

kt

0

------------- (2)

240 5.28 13.57 15.21 5.10 10.21 12.32 4.70 7.78 12.95 3.98 5.29 11.68

000 0.79 00.00 00.80 0.79 0.00 00.80 0.79 0.00 00.80 0.79 0.00 00.80

024 2.62 02.37 66.17 2.54 1.64 66.84 2.32 0.98 69.43 1.52 0.51 71.27

048 3.91 05.08 49.90 4.23 3.77 49.87 3.75 2.54 53.08 2.57 1.46 56.83

072 4.69 06.53 43.18 4.93 4.97 42.20 4.35 3.62 43.38 3.45 2.29 45.14

096 5.09 07.67 38.79 5.05 5.82 38.04 4.59 4.34 38.19 3.85 2.91 37.68

80 120 5.21 08.77 34.69 5.13 6.60 34.57 4.62 4.96 34.32 4.07 3.41 32.63

144 5.34 09.86 30.65 5.22 7.37 31.22 4.65 5.55 30.76 4.15 3.85 28.67

168 5.41 10.95 25.65 5.23 8.13 26.95 4.71 6.14 27.12 4.18 4.26 24.14

192 5.48 12.03 21.89 5.26 8.89 23.15 4.79 6.73 22.74 4.19 4.68 20.58

216 5.52 13.29 16.85 5.27 9.85 19.65 4.82 7.57 19.56 4.20 5.18 17.25

240 5.57 14.39 12.65 5.28 10.61 16.12 4.95 8.19 15.56 4.23 5.62 13.83

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The above equation represents concentration as a function of time.

To evaluate the model parameters and to relate the changes in substrate, cell mass and product concentrations, a Yield

coefficient is introduced. The above equation is cell mass kinetics.

B:Yield of growth:

The yield coefficients are related by the equation

SPY = XPSX YY ------------ (3)

Equation 3 indicates that the product formation rate is proportional to the cell and substrate utilization rate. In many

cases significant product formation occurs late in the log phase (approaching the stationary phase). One such model is

the Leudking- Piretkietic model. This model combines both growth-associated and non-growth associated

contributions:

xβrαr fxfp ------------ (4)

This two parameter kinetic expression has proved useful and versatile in fitting product formation data of many

different fermentation processes. The first term in the RHS represents the energy used for the growth and the second

term represents the energy used for maintenance. The above equation can be written as

xβdt

dxα

dt

dp ----------- (5)

Rearranging equation (5), integrating and inserting into Eq (2)

0

kt

s0

s

0 xx(t)αe1/xx1lnk

xβpp(t) -------- (6)

C: Substrate utilization kinetics

A part of the substrate is used for conversion to cell mass, a part used for product formation and another part for

maintenance. Therefore,

xkdt

dp

Y

1

dt

dx

Y

1

dt

dse

P/SX/S

-------(7)

Substituting equation (4) in (7)

xkxβdt

dxα

Y

1

dt

dx

Y

1

dt

dse

P/SX/S

------------- (8)

Simplifying and integrating

S=S0 + )1(1[ln{)1(1

1)1(. 0

0

0

kt

ktex

kexx

----- (9)

Equations(2), (6) and (9) describe the growth of cell mass, production of ethanol and substrate utilization respectively

with respect to time.

D: Estimation of kinetic parameters:

The above models contain the kinetic parameters k, α, β, γ and η. They should be evaluated using the experimental

data.

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Determination of k:

The cell mass production rate is given by the equation

kt

s

kt

exx

exx

1/1 0

0 -------------- (10)

Rearranging,

1/lnktx/x-1

x/xln 0

s

s xxs ------------- (11)

A plot of

tvsx/x-1

x/xln

s

s will yield the Riccati constant k

Estimation of Riccati constant k

Estimation of

In the stationary phase, dX/dt =0.and hence equation (5) gives,

s

phasestationary

βxdt

dp

-------- (12)

Estimation of Xs

dp dx

x

dt dt

Xs

Slope = k s

s

x/x-1

x/xln

t

Time

Cell mass

concentration

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The slope of the product concentrationcurve near the stationary phase will be a straight line. This value is divided by

the cell mass concentration at the stationary phase to get.

Estimation of

The product concentration is given by the equation (6). This equation is rearranged in the following way to determine.

0

kt

s0

s

0 xx(t)αe1/xx1lnk

xβpp(t) --------------(13)

kt

s0

s

0 e1/xx1lnk

xβpp(t)

0)( xtx

Estimation of growth constant

Estimation of

Using Yx/s, Yp/s and can be calculated using the following equation

;Y

1

Y

αγ

X/SP/S

---------------- (14)

Estimation of

Can be obtained using the following equation.

ekY

βη

P/S

-------- (15)

E: Model validation

Using the kinetic parameters obtained, concentrations of cell mass, product ethanol and substrate cellulose are

estimated as a function of time for each case. These predicted values are compared with experimentally determined

values. The closeness between these results should validate the model.

Error analysis

For each set of experimental data and for each of the variables x(t), P(t), C(t), the error between the predicted and

experimental values are calculated using the equations.

Slope =

Predicated Value - Experimental value

Error =

Experimental value

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For most of the cases, equation 2 predicts the cell mass concentration within 10%. Hence the Riccatitype equation is

valid and can be used with confidence. Equation 6predicts the concentration of ethanol with reasonable accuracy

(≤10%) for the pure culture and mixed culture.

The Fig 5 shows Comparison of experimental data and model predicted values for mixed cultures FO80%- NC20% .

Remaining ratio also like this and constants are presented in table II.

TABLE II

ESTIMATED VALUES OF MODEL PARAMETERS

Culture

K β Mixture Culture

Fusariumoxysporum Neurosporacrassa

80% 20% 0.087 0.97 0.0081

60% 40% 0.075 0.85 0.006

40% 60% 0.062 0.72 0.0049

20% 80% 0.045 0.65 0.004

ISC - 30 g/l

0

5

10

15

20

25

30

0 24 48 72 96 120 144 168 192 216 240

Time, h

Co

ncen

trati

on

, g

/lISC - 20 g/l

0

5

10

15

20

0 24 48 72 96 120 144 168 192 216 240

Time, h

Co

nce

ntr

atio

n,

g/l

C

ISC - 40 g/l

0

5

10

15

20

25

30

35

40

0 24 48 72 96 120 144 168 192 216 240

Time, h

Con

cent

rati

on, g

/l

ISC - 50 g/l

0

10

20

30

40

50

0 24 48 72 96 120 144 168 192 216 240

Time, h

Co

nce

ntr

atio

n,

g/l

ISC - 60 g/l

0

10

20

30

40

50

60

0 24 48 72 96 120 144 168 192 216 240

Time, h

Co

nce

ntr

atio

n,

g/l ISC - 70 g/l

0

10

20

30

40

50

60

70

0 24 48 72 96 120 144 168 192 216 240

Time, h

Co

nce

ntr

atio

n,

g/l

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V. CONCLUSION

The present investigation reveals certain important and useful facts. Both Fusariumoxysporum and Neurosporacrassa

yield ethanol in significant amounts and further studies can be carried out for commercial development of the process.

The effect of initial concentration of substrate is significant; the more initial concentration of cellulose the higher

production of ethanol. This is true for both the pure and mixed cultures. There is no substrate inhibition for the range

covered in this work.

For mixed cultures the rate of utilization of cellulose is high during the initial stage itself. However for pure cultures

the rate is slow initially due to lag phase. It may be that mixed culture do not have lag phase. Even though cell mass

production is high for wild culture, yield of alcohol is less indicating the formation of more by- products.

The results of the present research work can be summarized as follows:

1. Ethanol produced by wild culture Neurosporacrassa is least.

2. From the results of TableIII it is concluded that mixed culture with a composition of 80% Fusariumoxysporum

and 20% Neurosporacrassa can be used for maximum ethanol production.

TABLE: III

MAXIMUM ALCOHOL PRODUCTION BY THE DIFFERENT CULTURES

S. No ISC

(g/l) Culture Wild

Mixture

(FO80:20NC)

Mixture UV

(FO60:40NC)

01 20 FusariumoxysporumNeurosporacrass

a

6.48

5.3

9.62

6.09


a

6.85

5.69

10.11

6.99


a

7.06

6.4 10.69 7.86


a

7.82

6.79 11.72 8.72


a

8.35

7.32 12.32 9.63


a

8.97

7.78 13.57 10.48


a

10.0

8.03

14.39 11.32

Fig. 5Comparison of experimental data and model predicted values for mixed cultures FO80%- NC20%

ISC - 80 g/l

0

10

20

30

40

50

60

70

80

0 24 48 72 96 120 144 168 192 216 240

Time, h

Con

cent

ratio

n, g

/l

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Based on the kinetic models, equations have been developed for the determination cell mass, product and substrate

concentrations as a function of time. Using the experimental data, constants of these equations have been evaluated.

These mathematical relationships can be used with reasonable accuracy.

Mixed culture grows at a faster rate than the pure cultures till the stationary phase is reached. However the pure

cultures reach the stationary phase late and the final cell mass concentration is above that of mixed culture.

NOMENCLATURE

k - Riccati constant

P - Product concentration (g/l)

Po - Initial product concentration (g/l)

Pt - Product concentration at any time t, (g/l)

S - Substrate concentration, (g/l)

So - Initial substrate concentration, (g/l)

St - Substrate concentration at any time t, (g/l)

t - Time (h)

X - Cell mass concentration, (g/l)

Xo - Initial cell mass concentration, (g/l)

Xm - Maximum cell mass concentration, (g/l)

Xt - Cell mass concentration at any time t, (g/l)

Xs - Cell mass concentration in the Stationary phase, (g/l)

Y x/s - Yield coefficient (cell mass)

Y p/s - Yield coefficient (product)

Greek Symbols

- Growth Constant

- Maintenance Constant

- Specific growth rate, (h-1

)

m - Maximum specific growth rate, (h-1

)

γ - Constant η - Constant

Abbreviations X – Cell mass concentration, g/l

P - Concentration of Ethanol, ethanol, g/l

C- Concentration of cellulose, g/l

ISC- initial concentration of cellulose, g /l

FO – Fusariumoxysporum

NC – Neurosporacrassa

FO 20%- NC 80% -Fusariumoxysporum20% -Neurospora crassa-80%

FO 40%- NC 60% - Fusariumoxysporum40% -Neurospora crassa-60%

FO 60% -NC 40% -Fusariumoxysporum60% -Neurospora crassa-40%

FO 80% -NC 20% - Fusariumoxysporum80% -Neurospora crassa-20%

REFERENCES

[1] Bailey. J.E and D.F.Ollis,“Biochemical engineering fundamentals”2nd Edition, McGraw Hill, Newyork, 1986. .

[2] Christakapoulos. P, Macris. B.J and Kekos.D. “Direct fermentation of cellulose to ethanol by Furariumoxysporum.Enzyme Microbial

Technology” 11: 236-239, 1989.. [3] Deshpande.V, Keskar.S, Mishra C, Rao M. “Direct conversion of cellulose/ hemicellulose to ethanol by Neurosporacrassa.Echnology”8: 149–

152, 1986.

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ISSN (Print) : 2347 - 6710






[4] Ikram.U.H, Hamad.A and Sikander.A “Selection of Hyper Secretion Mutant of Bacillus icheniformis for Alpha amylase production”,

proceedinds of international conference 1:169 – 173, 2003. [5] Kadam.K.L, Robert J. Wooley, Andy Aden, Quang A, Nguyen Mark A. Yancey and Francis M. Ferraro“Softwood forest thinning as a biomass

source for ethanol production”, A feasibility study for California Biotechnology 16: 947-957, 2000. [6] Lynd. LR, Weimer. PJ, Zyl. WH and Pretorius. IS.“Microbial cellulose utilization”, Fundamentals and biotechnology Microbial Rev 66:506–

577, 2002..

[7] Mielenz. JR. “Ethanol production from biomass: technology and commercialization status”,CurrOpinMicrobiol 4:324–329, 2001.

[8] OS. D, Macris. BJ andChristakopoulos. P, “ Purification and characterization of NAD+ dependent xylitol dehydrogenase from Fusariumoxysporum “, Biotech Lett 24:2089–2092, 2002.

[9] Panagiotou. G, Kekos. D, Macris. BJ and Christakopoulos.P., “The influence of aeration and external electron acceptors on xylose fermentation

by Fusariumoxysporum.Isolation of two NADPH-depentent d-xylose reductases”, Journal BiosciBioeng 97:299–304, 2004. [10 ]akopoulos.P, Jens Nielsena and Lisbeth Olssona.,“Intracellular metabolite profiling of Fusariumoxysporumconverting glucose to ethanol”,

Journal of Biotechnology 115: 425–434, 2005a.

[11] Panagiotou. G.A, Christakopoulos.P and Olssona.L.“Simultaneoussaccharification and fermentation of cellulose by Fusariumth characteristics and metabolite profiling” Enzyme and Microbial Technology ,36:693–699, 2005b.

[12] A.B, Christakopoulos. P, Villas-Boasa.S.G and Olssona.L., “Fermentation performance and intracellular metabolite profiling of

Fusariumoxysporumcultivated on a glucose–xylose mixture”,Enzyme and Microbial Technology 36:100–106, 2005c. [13] Sadasivam. S and Manickam. A“BiochemicalMethods”,second edition, New age Internal Publishers, New Delhi, 1996.

[14]Stevenson. D.M and Weimer P.J“Isolation and characterization of a Trichoderma strain capable of fermenting cellulose to ethanol”, Applied

Microbial Biotechnology,59: 721 – 726, 2002. [15]Zhou S and Ingram LO.“Simultaneous saccharification and fermentation of amorphous cellulose to ethanol by recombinant

KlebsiellaoxytocaSZ21 without supplemental cellulose”, Biotechnology Letter, 23:1455–1462, 2001.

[16]HaoZhang ,Haizhen Mo and Haijuan Nan, “Bioconversion of cellulose to bioalcohol”, International Conference on Agricultural and Biosystems Engineering ,Advances in Biomedical Engineering Vols. 1-2, 2011.

[17]Priyachandrakant and Y.S Bisaria,“ Simultaneous bioconversion of cellulose and hemicellulose to ethanol” ,Department of Biochemical

Engineering and Biotechnology, Indian Institute of Technology, HauzKhas, New Delhi, India, Vol. 18, No. 4 , Pages 295-331, 1998. [18]Ming-Jun Zhu , Zhi-Sheng Zhu and Xu-Hui Li , “ Bioconversion of paper sludge with low cellulosic content to ethanol by separate hydrolysis

and fermentation”, African Journal of Biotechnology, Vol. 10(66), pp. 15072-15083, 2011.

[19].Miguel A. Medina-Morales, J.C. Contreras-Esquivel , et.al., “Enzymatic Bioconversion of Agave Leaves Fiber Hydrolysis Using Plackett-Burman Design”, American Journal of Agricultural and Biological Sciences, 6 (4): 480-485, 2011.

[20]Sonil Nanda, Ajay K. Dalai &Janusz A. Kozinski ,“Butanol and ethanol production from lingo cellulosic feedstock: biomass pretreatment and bioconversion”,Energy science and engineering ,Vol2 issue 3, 24 , July 2014.

[21] Lezinou.V, Christakopoulos.P, Kekos.D. andMacris. B.J “Simultaneous saccharifieation and fermentation of sweet sorghum carbohydrates to

ethanol in a fed-batch process”, Biotechnology Letter16:983-988, 1994.

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